*Hemocytometer or disposable slides for the automated counter or manual counter

*Hemocytometer or disposable slides for the automated counter or manual counter

-

*50 mL Accuspin tubes, radiation sterilized. (Sigma #A-2055)

*50 mL Accuspin tubes, radiation sterilized. (Sigma #A-2055)

*Sterile 5mL polypropylene round-bottom tubes

*Sterile 5mL polypropylene round-bottom tubes

Line 48:

Line 40:

# Add up to 30mL of diluted blood to the accuspin tube.

# Add up to 30mL of diluted blood to the accuspin tube.

# Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).

# Centrifuge at 500 g for 30 minutes at room temperature (slow acceleration, deceleration off to ensure no disruption of the density gradient).

-

# Using a sterile transfer pipette, aspirate the buffy coat (peripheral blood mononuclear cells [PBMCs]) into a new 50mL conical tube. (Avoid aspirating the Ficoll.) Add PBS to bring to a minimum of 2x the volume, inverting up and down to mix.

# In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined.

# In order to determine the volume to use for resuspending the PBMCs after the wash, the total number of cells in the sample must be determined.

-

# Carefully introduce 10 μL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter. Calculate the number of cells, taking into account the dilution factors and sample volume used. Or place 20 μL of the stained cells onto a disposable slide and count using the automated cell counter.

+

# Carefully introduce 10 μL of the stained cells into the notch of a hemocytometer and record cell counts using a hand-held counter.

-

# After centrifugation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with AIM-V at 4 x 10<sup>6</sup>/ml in 15 ml PP conical tube.

+

# Calculate the number of cells, taking into account the dilution factors and sample volume used. Or place 20 μL of the stained cells onto a disposable slide and count using the automated cell counter.

+

# After centrifugation is completed, aspirate and discard the supernatant.

+

# Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs with AIM-V at 4 x 10<sup>6</sup>/ml in 15 ml PP conical tube.

==PBMC Stimulation==

==PBMC Stimulation==

'''(Sterile Technique)'''

'''(Sterile Technique)'''

-

# Remove stimulants from the freezer and thaw.

+

# Remove Musm1 from the freezer and thaw.

-

# Label 5 mL sterile polypropylene tubes, each well with the appropriate stimulant condition, '''ordered by priority''' (for cases where there are insufficient cells to test all stimulants).

+

# Centrifuge the Musm1 briefly in the small centrifuge.

+

# Label 5 mL sterile polypropylene tubes with the appropriate stimulant condition, '''ordered by priority''' (for cases where there are insufficient cells to test all stimulants).

## Musm1 (allergen): @ 200 μg/mL purified Musm1 protein in Aim-V.

## Musm1 (allergen): @ 200 μg/mL purified Musm1 protein in Aim-V.

## AIM-V (negative control): AIM-V medium alone.

## AIM-V (negative control): AIM-V medium alone.

## Beads (positive control): 1μg/mL anti-CD3/CD28 beads.

## Beads (positive control): 1μg/mL anti-CD3/CD28 beads.

# Prepare solutions for each stimulant condition as follows:

# Prepare solutions for each stimulant condition as follows:

-

#* 200 μg/mL MusM1 in AIM-V: place 450 μL of AIM-V in the appropriately labeled tube and add 50 μL of allergen (MusM1). Then add 500 μL of cell suspension. Pipette up and down.

+

#* 10 μg/mL MusM1 in AIM-V:

-

#* AIM-V medium alone: place 500 μL of AIM-V in the appropriately labeled tube and add 500 μL of cell suspension. Pipette up and down.

#: Note: There will be two aliquots of supernatant collected for each culture. These should be stored in two separate cluster tube boxes and shipped on separate days to minimize the risk of loss during storage/ shipment.

# Label two cluster tubes for each culture tube as follows:

# Label two cluster tubes for each culture tube as follows:

## Specimen ID

## Specimen ID

Line 90:

Line 99:

### Stimulant 2 = AIM-V

### Stimulant 2 = AIM-V

### Stimulant 3 = Beads

### Stimulant 3 = Beads

-

#: For each stimulant, using a 1000μL pipette tip, transfer 400 μL of supernatant from the culture tube into each corresponding cluster tube in the cluster rack. Be careful not to disturb the cell pellets.

+

# For each stimulant, using a 1000μL pipette tip, transfer 380 μL of supernatant from the culture tube into each corresponding cluster tube in the cluster rack.

-

#: Note: There will be two aliquots of supernatant collected for each culture. These should be stored in two separate cluster tube boxes and shipped on separate days to minimize the risk of loss during storage/ shipment.

+

#: Take care to not disturb the cell pellets.

# Cap the cluster tubes and store in the –80°C freezer.

# Cap the cluster tubes and store in the –80°C freezer.

Line 97:

Line 106:

'''(non-sterile technique)'''

'''(non-sterile technique)'''

-

# Add 1mL of staining buffer to the cell pellet of each culture tube, vortex on low.

# Centrifuge cells at 400 g for 5 minutes at 4°C. Please take special care to minimize the cell pellet disturbing or lost. Use a 1000 μL pipetman to aspirate most of the supernatant removing the last ~100 μL with a 200 μL pipetman. Make sure that only filtered pre-sterilized tips are being used from this step and later.

#: Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil

#: Note: good for one month after adding β-ME, store in the dark and wrapped in aluminum foil

-

# Use a filtered pipette tip to add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully. Vortex each Eppendorf tube twice for 15 seconds each time and spin briefly at 1,000g for few seconds to collect all liquid at the bottom of the tube. Transfer to labeled cryovial tube.

+

# Add 1 mL of staining buffer to the cell pellet of each culture tube, vortex on low.

+

# Centrifuge cells at 400 g for 5 minutes at RT. Please take special care to minimize disturbing the cell pellet.

+

# Aspirate supernatant down to line at tube curvature (~50 μL ) using a pasteur pipette with unfiltered 200 μL pipette tip.

+

# Add 300 μL of the RLT buffer containing β-mercaptoethanol and resuspend cells by pipeting up and down 10 times, rinsing walls of tubes carefully.

Collection of Supernatants

Visually inspect the cell cultures and make a note of variations in pellet size, culture media color, signs of contamination.

Mix the cells carefully by pipetting up and down to resuspend cells in the tube and evenly mix any secreted cytokine.

Take a 10 μL aliquot of each culture to inspect under the microscope.

Mix cell aliquot with equal volume of Turks solution and load 10 μL on a hemacytometer.

Note variations in cell density, obvious contamination.

Centrifuge tubes at 400g for 5 minutes at 25°C.

Obtain a cluster tube rack for the storage of supernatants.

Note: There will be two aliquots of supernatant collected for each culture. These should be stored in two separate cluster tube boxes and shipped on separate days to minimize the risk of loss during storage/ shipment.

Label two cluster tubes for each culture tube as follows:

Specimen ID

Date

Supernatants - 48 hour

Stimulant condition 1-3

Stimulant 1 = MusM1

Stimulant 2 = AIM-V

Stimulant 3 = Beads

For each stimulant, using a 1000μL pipette tip, transfer 380 μL of supernatant from the culture tube into each corresponding cluster tube in the cluster rack.